The disclosure relates to synthetic oligonucleotides that are unique in that they are RNA molecules that have the capacity to form a hydrogel. Also disclosed are DNA oligonucleotides that encode the RNA oligos so that the oligos can be prepared using in vitro transcription. The disclosure further pertains to pharmaceutical compositions comprising these hydrogels.
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CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation of PCT/US2015/054942 filed Oct. 9, 2015 and published on Apr. 14, 2016 as WO 2016/05792, which claims the priority of U.S. provisional application No. 62/061,757 filed Oct. 9, 2014; the contents of each are hereby incorporated by reference in their entirety into the present disclosure.
STATEMENT OF RIGHTS UNDER FEDERALLY-SPONSORED RESEARCH
This invention was made with government support under grant number W81XWH-09-1-0568 awarded by the U.S. Army Medical Research Materiel Command. The government has certain rights in the invention.
This application contains a Sequence Listing, created on Oct. 7, 2015 and modified on Apr. 23, 2018; the file, in ASCII format, is designated 0794151A_Sequence Listing_ST25.txt and is 6.47 KB in size. The file is hereby incorporated by reference in its entirety into the application.
The disclosure relates to hydrogels and in particular, RNA hydrogels.
BACKGROUND OF THE INVENTION
Hydrogels are water-saturated turgid materials and can be used for a wide range of applications such as tissue engineering scaffolds and drug delivery vehicles. Biomolecules, such as lipids, peptides, proteins, polysaccharides, and deoxyribonucleic acid (DNA), but not ribonucleic acid (RNA), have been found to form hydrogels.
SUMMARY OF THE INVENTION
In one aspect, the disclosure relates to a synthetic RNA oligonucleotide comprising the secondary structure of:
In one embodiment, the synthetic oligonucleotide consists of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2. These oligonucleotides form hydrogels.
In a related aspect, the disclosure relates to a synthetic oligonucleotide that encodes an RNA with the nucleotide sequence of SEQ ID NO: 1.
In yet another aspect, the disclosure relates to a synthetic oligonucleotide that encodes an RNA with the nucleotide sequence of SEQ ID NO: 2.
In yet another related aspect, the disclosure relates to a pharmaceutical composition comprising a synthetic oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
BRIEF DESCRIPTION OF THE DRAWINGS
DETAILED DESCRIPTION OF THE INVENTION
All patents, publications, applications and other references cited herein are hereby incorporated by reference into the present application. Methodology used in developing the present invention are well known to those of skill in the art and are described, for example, in Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; DNA Cloning: A Practical Approach, Volumes I and II, 1985 (D. N. Glover ed.); Oligonucleotide Synthesis, 1984 (M. L. Gait ed.), the contents of which are hereby incorporated by reference. In the description that follows, certain conventions will be followed as regards the usage of terminology.
Hydrogels are supramolecular assemblies hosting aqueous media with nearly identical solute transport properties of liquid and also mechanical properties of solid1,2. These properties make hydrogels uniquely useful, especially for biomedical applications, such as drug delivery vehicles3-7.
Hydrogels have attracted considerable attention as promising biomaterials for various biotechnological and biomedical applications because of their high water content, favorable structural features and biocompatibility.
With the establishment of the first synthetic hydrogels by Wichterle and Lim in 195431, hydrogel technologies have become applicable to hygienic products32, agriculture33, drug delivery systems32, 34, sealing32, coal dewatering35, artificial snow32, food additives36, pharmaceuticals37, biomedical applications36, 39 tissue engineering and regenerative medicines40,41, diagnostics42, wound dressing43, separation of biomolecules or cells44 and barrier materials to regulate biological adhesions45, and biosensor46.
Applications of particular interest in the present disclosure are those that include tissue engineering, drug delivery platforms, and cell culture scaffolds. Biocompatible hydrogels establish and regulate the mechanical properties of cells and tissues and thus serve as lubricants in joints or on epithelial surfaces. These gels can also serve as selective filters or selective barriers for a broader range of permeability control. In addition, hydrogels loaded with drugs can be used for wound dressing.
To date, all naturally occurring biomolecules are known capable of forming hydrogels or biocompatible hydrogels3-6,8, except RNA. As the other type of nucleic acids, DNA can readily form hydrogels since the double stranded nature of DNA chains allows relatively intuitive design of network structures by base pairing interactions9-11. Specifically, short linear double-stranded DNA with designed sticky ends are synthesized as monomeric building blocks for hydrogels. In addition, these stick ends can be covalently linked as catalyzed by DNA ligases to join multibranched DNA monomers10,11.
In contrast, RNAs are generally single stranded; they can form intra-strand double helixes and adopt complex tertiary structures, based on Watson-Crick base paring (guanine-cytosine or G-C and adenine-uracil or A-U), noncanonical base pairing (e.g., G-U or A-A) and complex tertiary interactions, such as base stacking, kissing loops and pseudoknots12,13. As a result, RNA exhibits complex biological functions such as catalysis (please see The RNA World, 3rd ed.; Gesteland R. F., Cech T. R., Atkins J. F., Eds.; Cold Spring Harbor Laboratory (CSHL) Press: Cold Spring Harbor, N.Y., 2013.), inhibition14 and regulation of gene expression15. However, RNA has not been known previously to either possess functional parts, similar to “sticky ends” in DNA hydrogels, or modular sequence segments as designer elements for network assembly through intermolecular interactions to form hydrogels. Here we describe an RNA whose sequence contains two unique sequence segments or motifs. The RNA can form hydrogel because of these sequence motifs. We show that these motifs are responsible for RNA self-assemblies to form a hydrogel network structure.
In one embodiment, an RNA molecule that can form a hydrogel has the nucleotide sequence of SEQ ID NO: 1:
Initially, an RNA aptamer was selected from a library of ˜1014 sequences using SELEX, an in vitro evolution experiment16,17 We initially aimed to find an RNA aptamer capable of positively enhancing AMPA receptor response to glutamate, the endogenous neurotransmitter in the central nervous system, thereby increasing the amplitude and/or the duration of synaptic responses in vivo. Positive modulation of AMPA receptors has long been suggested to affect cognition18,19. For instance, depolarization at dendritic spines (i.e., small appendages of dendrites) via AMPA channels is linked to the induction of long-term potentiation (LTP), a presumed substrate of memory20. Studies of the interaction between glutamatergic and monoaminergic systems suggest the link of a reduced glutamatergic transmission to certain cognitive disorders, such as schizophrenia and Parkinson's disease21. Potentiators of AMPA receptors are drug candidates in a treatment of cognitive disorders22. In our experiment, the in vitro selection target was GluA2, a key AMPA receptor subunit23. The receptors were transiently expressed in human embryonic kidney (HEK-293) cells, and the lipid membrane fragments that contained GluA2 receptors were used as the selection target24.
After a 12-round selection, an RNA, which we term “CZ aptamer” (the predicted secondary structure using MFold are shown in
Surprisingly, CZ aptamer turned into an elastic solution state in a standard enzymatic transcription reaction mixture in just a few hours after transcription reaction was initiated. Motivated by this finding, a 237-nt 2CZ RNA whose sequence was essentially a double repeat of the CZ aptamer (see its predicted secondary structure in
Similarly to the CZ aptamer, 2CZ aptamer also formed an elastic solution state. To confirm that the RNA was responsible for the gel state, we purified the 2CZ RNA (lane ‘A’ of the right panel in
To further verify that 2CZ RNA could form hydrogel, we lyophilized the sample. Lyophilization of the RNA solution left sponge-like textured material in the tube with nearly similar volume before dehydration, indicating the RNA chains span all the solution volume (right panel,
The viscoelastic properties of this hydrogel was investigated by employing a temperature sweep rheology test under the condition of small amplitude oscillation (1 rad/s) and strain (1%) at a heating/cooling rate of 0.1° C./min (
Next we carried out small angle X-ray scattering (SAXS) experiments to investigate spatial correlation of RNA hydrogel structures in solutions (
Because the CZ RNA aptamer we discovered is uniquely capable of self-assembling to form hydrogel, a single RNA molecule must contain at least two unique sequence segments. These two sequence segments will enable a single RNA molecule as a “monomer” to bind non-covalently with the same sequence segments of at least two other RNA molecules so that RNA molecules could “grow” longer to form suprastructures, as the basis of hydrogel matrix. These unique sequence segments, possibly in motif forms, for inter-molecular interactions should be available after an RNA first folds intra-molecularly. To investigate the existence and the nature of these unique sequences, we first used MFold, an RNA secondary structure prediction program, to examine the CZ RNA “monomer” (we reasoned that a 2CZ RNA, as in
We also identified two critical residues or GC adjacent to each other in the middle of motif 1 for gelation (
The mutation experiment and analysis have shown that both motifs 1 and 2 in a CZ RNA molecule are required to form RNA network structures as the basis of the hydrogel. There are at least two interesting conclusions to be drawn from this finding. Firstly, each motif should serve as an “arm” in its own direction to extend its non-covalent contact with the same motif but from another RNA molecule. Together, the CZ RNA aptamer with the two motifs is like an “elbow joint” as the basic constructional unit to build a complex RNA network matrix, minimally thought x-y direction. In this sense, the RNA sequence of a motif should not be special. To prove this point, we engineered two special CZ RNA mutants in that one contains two motifs 1, whereas the other contains two motifs 2. The mutant with two motifs 1, which uses ACGU as canonical Watson-Crick base pairing residues, as expected, did form a gel, while the mutant with two motifs 2 didn't. Again, this result is consistent with our conclusion that motif 1/motif 1 interaction is stronger than motif 2/motif2 interaction in forming an RNA network matrix for hydrogel. Therefore, our study in essence has enabled us to identify an architectural principle by which an extensive RNA network can self-assemble through inter-molecular interactions using these motifs. Second, other types of interactions may be also involved in the network folding and interactions of these RNAs, in addition to strong canonical Watson-Crick base pairing through the two corresponding two motifs (
The present disclosure also encompasses RNA oligonucleotides in which one or more nucleotide substitutions has been made to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as long as the motif structure required for hydrogel formation as described above is maintained. Examples of additional RNA oligonucleotides examined for formation of hydrogels:
Of these, only d5.CTZ1214 and d6.CTZ1214 have not yet been shown to form a gel.
These examples illustrate that an RNA molecule with specific sequence motifs that can interact with other RNA molecules in sufficient concentration can form a hydrogel by self-assembling into a polymeric network structure. Similar to other types of biocompatible hydrogels, RNA hydrogels may therefore be explored for similar applications such as drug delivery (in our case, the RNA itself is a potentiating aptamer for AMPA receptors). It should be noted that for these biological application, RNA hydrogels with improved mechanical and functional properties, such as a prolonged drug release controlled by RNA hydrogel degradation, could be obtained through chemical cross-linking to change mesh size, as an example. The mesh size can be readily achieved by the use of cross-linker density. The fact that 2CZ RNA aptamer, which now contains an extra stem-loop in between the two “monomeric” CZ sequence, is fully capable of forming hydrogel further suggests that additional sequences or motifs, irrelevant to hydrogel formation, can be designed as a functional platform to link other RNAs or “code” for a new function with the hydrogel. For example, this stem-loop can be replaced with an aptamer for molecular recognition and sensing.
Synthetic RNA oligonucleotides of the disclosure may be prepared by any method known to those of skill in the art, including chemical synthesis, isolation from a nucleic acid library or by recombinant technology. In one embodiment, the method of preparing a nucleic acid ligand of the invention begins by identifying nucleic acid ligands from a candidate mixture of nucleic acids by Systemic Evolution of Ligands by Exponential Enrichment (SELEX), or a variation thereof, which is a commonly used method of identifying nucleic acid ligands that bind to a target from a candidate mixture of nucleic acids.
Cell Culture and Transient Receptor Expression
The original cDNAs encoding rat GluA2Qflip AMPA receptor was kindly provided by Steve Heinemann. The GluA2Qflip receptors were transiently expressed in human embryonic kidney (HEK-2935) cell. HEK-2935 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin in a 37° C., 5% CO2, humidified incubator. The DNA plasmids encoding green fluorescent protein and large T-antigen were cotransfected in HEK-293S cells (18). Transfected cells were used for recording 48 hour after transfection. For the in vitro selection, the transfected cells were harvested 48 hours after transfection, and the membrane fragment that contained the GluA2Qflip receptor was prepared as described (19).
In Vitro Selection
The preparation of the RNA library and the protocol of running the in vitro evolution selection were described previously (19). For binding in the initial round of selection, the RNA library with ˜1015 random sequences was dissolved in the extracellular buffer, which contained (in mM) 150 NaCl, 3 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES (pH 7.4). The final concentration of membrane-bound receptor in the binding mix was 8 nM, as determined by [3H]AMPA binding. The membrane-bound receptor was exposed to 0.4 mM of cyclothiozide (CTZ) for 5 min and then to 50 mM of glutamate for 5 min. The mixture was then mixed with the RNA library. The mixture of the RNA library and the receptor was incubated at a thermo-cycle as follow: 22° C./40 min→37° C./10 min→22° C./20 min→(37° C./5 min→22° C./20 min)×2. After the thermal cycle, the mixture were filtered through a 0.45 μm nitrocellulose filter (Millipore, HAWP02500). The filter was washed with 15 ml of 1× extracellular buffer 3 times. The filter was transferred to a fresh tube containing 0.5 ml of 10 mM Tris-HCl with 8 M urea. The tube was incubated at 95° C. for 5 min and extracted with phenol/chloroform/isoamyl alcohol (24:24:1). The RNA in the water phase was precipitated by ethanol, air-dried, and dissolved in H2O. The reverse transcription and PCR were described as previously (19). Starting from cycle 5, the RNA library was mixed with 10 mg of cell fragments, which prepared from the HEK293S cells transient transfected with TAg plasmid, and incubated at 22° C. for 20 min. The mixture was passed through a nitrocellulose filter. The solution filtered through was collected and used as the RNA library for the binding reaction. The cell membrane fragments without GluA2 was used to remove non-target binding RNA from the library. At the end of the 12th selection round, the DNA pools from rounds 10, and 12 were separately cloned into the pGEM-T easy vector (Invitrogen) for sequencing.
In one embodiment, preparation of RNA hydrogel according to the disclosure is by RNA in vitro transcription in accordance with methods known in the art using DNA templates for in vitro transcription by PCR amplification. Following transcription, the presence of a hydrogel is generally confirmed by visual inspection of the transcription reaction mixture.
DNA nucleotide sequences for in vitro transcription are as shown below.
RNA In Vitro Transcription
The RNA in vitro transcription reaction contained 0.3 μM DNA template, 10 mM of each NTPs, 50 ng/μl of T7 RNA polymerase, 0.005 unit/μl of pyrophosphatase, 25 mM of MgCl2, 10 mM DTT, 2 mM spermidine, and 50 mM HEPES (pH 7.5). The transcription mixture was incubated at 37° C. overnight and then kept in a 4° C. refrigerator.
RNA PAGE Purification
A tubular PAGE column system (Bio-Rad Prepcell 491) was used to purify RNA from the transcription mixture. The detailed method has been described previously (cite HPLC paper). The polyacrilamide gel column was formed by 80 ml of 12% acrylamide/bis-acrylamide (37.5:1) solution in 1×Tris-Borate-EDTA (TBE) buffer containing 8 M urea. For each run, about 1 ml of the transcription mixture was mixed with 1 ml of gel loading buffer II (Biorad), which contained 95% of formamide. The RNA transcription sample with loading buffer was incubated at 95° C. for 5 min and loaded on the surface of the PAGE column. The electrophoresis was run at 250 V for 10 hours. A peristaltic pump was used to regulate the mobile phase or 1×TBE buffer at 1 ml/minute flow rate. The elution of the RNA sample was monitored by a UV detector and collected in a fraction collector (BioFrac, Bio-Rad) at 1.5 ml/fraction. The fractions were then pooled based on the chromatography trace and concentrated in an Amicon filtration centrifuge tube (Millipore). The TBE buffer in the eluted samples was exchanged with 25 mM HEPES buffer by spinning in an Amicon filtration tube; the same procedure was repeated two more times. The concentration of the collected sample was determined by a Nanodrop 1000 spectrophotometer (Thermal Fisher Scientific).
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1. A synthetic RNA oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, 8, 9, 15 or 16.
2. A synthetic RNA oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 1, 8, 9, 15 or 16.
3. A synthetic RNA oligonucleotide comprising tandem repeats of the nucleotide sequence of SEQ ID NO: 1, 8, 9, 15 or 16.
4. The synthetic oligonucleotide of claim 3 consisting of the nucleotide sequence of SEQ ID NO: 2.
5. The synthetic oligonucleotide of claim 3 comprising the nucleotide sequence of SEQ ID NO: 2.
6. The synthetic oligonucleotide of claim 3, where said tandem repeats are separated by a linking/intervening nucleotide sequence.
7. A hydrogel comprising the synthetic oligonucleotide of claim 1.
8. A synthetic oligonucleotide that encodes an RNA of claim 1.
9. A synthetic oligonucleotide of claim 8, wherein the oligonucleotide consists of the nucleotide sequence of SEQ ID NO: 3.
10. The synthetic oligonucleotide of claim 8, wherein the oligonucleotide consists of the nucleotide sequence of SEQ ID NO: 4.
11. A pharmaceutical composition comprising a synthetic oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, 2, 8, 9, 15 or 16.
12. A pharmaceutical composition comprising a hydrogel comprising an oligonucleotide with the nucleotide sequence of SEQ ID NO: 1, 2, 8, 9, 15 or 16.
U.S. Patent Documents
|20080138408||June 12, 2008||Venkatesh|
Filed: Apr 3, 2017
Date of Patent: Oct 23, 2018
Patent Publication Number: 20170335319
Assignee: THE RESEARCH FOUNDATION FOR THE STATE UNIVERSITY OF NEW YORK (Albany, NY)
Inventors: Li Niu (Loudonville, NY), Zhen Huang (Latham, NY)
Primary Examiner: Ekaterina Poliakova-Georgantas
Application Number: 15/477,498
International Classification: C12N 15/11 (20060101); C12N 15/113 (20100101); G01N 33/545 (20060101); C12Q 1/68 (20180101); A61K 38/00 (20060101);